Cell type specific transcription is achieved through the integration of regulatory inputs from multiple classes of cis-regulatory elements, such as enhancers, silencers and insulators. Insulators are the class of cis-regulatory elements that establish transcriptional fidelity through the formation of topological domains, which prevent enhancers and silencers from interacting with non-target promoters. Defects in insulator function have been identified in cancers, imprinting syndromes, and repeat expansion diseases, highlighting the importance of insulator function in transcriptional regulation. Mounting evidence suggests that insulator-binding proteins are multi-functional regulators, serving as transcriptiona activators and repressors and contributing to non- transcriptional functions, such as DNA replication. The challenge in the field is to determine how a single insulator protein mediates such diverse regulation and whether a role in chromosome architecture unifies all functions. We address this question through studies of the Drosophila Suppressor of Hairy-wing insulator protein, a multi-functional twelve zinc finger DNA binding protein. Three aims are proposed. First, we will determine how Su(Hw) establishes insulator function. Second, we will define how genomic context of an Su(Hw) binding sites influences Su(Hw) transcriptional functions. Third, we will establish the requirements for Su(Hw) in chromosome structure and DNA replication. These investigations will elucidate how a single insulator-binding protein confers multiple regulatory outputs. Information gained from these studies will establish a paradigm for understanding mechanisms involved in transcriptional regulation by the human multivalent insulator-binding protein, CCCTC binding factor.